The long-term objective of this proposal is to unveil the pleiotropic activity of tissue inhibitor of metalloproteinases (TIMP)-1 during breast cancer progression. Emerging clinical evidence indicates that TIMP-1 is a potent predictor of poor prognosis in breast cancer patients. However, the mechanisms of action of TIMP-1 in breast epithelial cells are not completely understood. In addition to its classical activity as an inhibitor of metalloproteinase (MP) activity, accumulating evidence indicates that TIMP-1 can also promote cell survival independently of MP inhibition. During the past funding period, we conclusively showed that TIMP-1 is a potent inhibitor of both intrinsic and extrinsic apoptosis independent of its MP inhibitory activity. Importantly, our recent study identified the tetraspanin member CD63 as the first TIMP-1 binding cell surface protein which modulates the integrin 1 survival pathway. TIMP-1 activation of the CD63/integrin signaling complex implies potentially diverse effects of TIMP-1 on many cellular processes. Preliminary data show that TIMP-1 also inhibits lumen formation and apoptosis during morphogenesis of non-malignant breast epithelial MCF10A acini in three-dimensional matrigel cultures. Moreover, TIMP-1 interaction with CD63 induces an epithelial mesenchymal transition (EMT) via upregulation of the EMT master transcription factor Twist, known to promote cancer progression. Structurally, TIMP-1 binds to CD63 via its non-MP inhibitory C-terminal domain as it does to latent pro-MMP-9. This suggests a novel mechanism of TIMP-1 regulation at the cell surface, in which the availability of pro-MMP-9 in the pericellular space may regulate TIMP-1/CD63 interactions. Based on our previous and preliminary studies, we hypothesize that TIMP-1/CD63 activation of integrin 1/a signaling complex activates focal adhesion (FAK) and phosphatidylinositol 3-kinase (PI3K) leading to cell survival, increased Twist expression and EMT during the early stages of tumor progression and metastasis. To test our hypothesis, we propose (1) to investigate the molecular mechanisms by which TIMP-1 and CD63 regulate integrin heterodimer functions, (2) to investigate TIMP-1/CD63-mediated intracellular signal transduction pathways for the regulation of cell survival and EMT, (3) to establish the role of pro-MMP-9 in the regulation of TIMP-1/CD63-mediated cell survival, and (4) to examine the in vivo roles of TIMP-1 during breast cancer progression. Accomplishment of the proposed studies will contribute to the collective endeavor to understand the multi functions of TIMP-1 during breast cancer progression. This information may also be useful for the development of novel approaches for targeting TIMP-1's signaling activity versus its MP inhibitory activity. PUBLIC HEALTH RELEVANCE: Tissue inhibitor of metalloproteinases (TIMP)-1, a natural inhibitor of matrix metalloproteinases (MPs), has been shown to inhibit tumor cell invasion in vitro and tumor progression in animal models of later stages of tumor metastasis. However, emerging clinical evidence indicates that TIMP-1 is a potent predictor of poor prognosis in breast cancer patients, an unexpected observation in light of these previously reported studies. Accumulating evidence provided by many investigators including us indicates that TIMP-1 can also promote cell survival independently of its MP inhibition. Interestingly, the preliminary data show that TIMP-1 also induces epithelial mesenchymal transition (EMT) phenotype in breast epithelial cells. The goals of the current application are to unveil molecular mechanisms by which TIMP-1 regulates cell survival and EMT, and to examine its potential oncogenic activity in an animal model of the early stages of breast cancer progression. Accomplishment of the proposed studies will contribute to the collective endeavor to understand the multiple functions of TIMP-1 during breast cancer progression. This information may also be useful for the development of novel approaches for targeting TIMP-1's signaling activity versus its MP inhibitory activity.